Automated transient image cytometry

ABSTRACT

A method, system, and instrument for automatically measuring transient activity in cells uses image time sequences to identify transients in cells. Preferably, the transient activity is stimulated or provoked in synchronism with acquisition of the image time sequences. A cell mask is applied to each image of an image time sequence in order to localize the transient activity with respect to each cell. Localization enables cell-by-cell analysis of properties of the transient activity.

PRIORITY RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/454,217, filed May 13, 2009 for “Automated Transient ImageCytometry”, which will issue as U.S. Pat. No. 8,385,624 on Feb. 26,2013.

This Application contains subject matter related to the subject matterof the U.S. patent application Ser. No. 11/285,691, filed Nov. 21, 2005,and published as US 2008/0144895 A1 on Jun. 19, 2008, and to the PCTcounterpart, PCT/2005/044936, filed Nov. 17, 2006, and published as WO2007/061971 A2 on May 31, 2007.

STATEMENT OF GOVERNMENT INTEREST

The invention described herein were made in part with government supportunder Grant No. 1R42HL086076 awarded by the National Institutes ofHealth. The United States Government has certain rights in theinvention.

BACKGROUND

The field is processes, systems, and instruments for automaticallymeasuring transient activity in cells using image sequences. Morespecifically, the field includes use of image cytometry to automaticallymeasure calcium transients in cardiomyocytes.

Automated image cytometry is increasingly used in high content screening(HCS) to automatically measure characteristics of objects in magnifiedimages of cellular material. Typically, an automated image cytometrysystem includes tools to perform analysis of images and analysis of dataacquired from the images. Image analysis may include staining materialwith one or more dyes, each selected to bind with a particular structureor component of the material. For example samples in a multi-wellscreening experiment related to obesity may be stained with one dye thatbinds specifically with cell nuclei, a second dye that bindsspecifically with cell cytoplasm, and a third dye that binds with lipiddroplets. Magnified images of well contents may be selectively processedaccording to color in order to classify image objects such as nuclei,cell perimeters, and lipid droplets. Once classification is complete,data may be extracted from the classification results in order toquantify various properties of the objects. See, for example the relatedUS publication 2008/0144895 A1.

Heart failure due to hypertension, infarction, or other factors, is aleading killer of men and women in modern society and involves, at itsbase, a debilitating loss of cardiomyocytes. Encouraging recent studiesindicate the feasibility of regenerating lost cardiomyocytes, forexample by transplanting embryonic stem cell-derived cardiac myocytes(ESCMs) or mobilizing resident stem cells. To realize the potential ofstem-cell based therapies, it would be extremely beneficial to developtechnology and instruments for high throughput, high content screening(HCS) of drugs and genes for their ability to stimulate the formation offunctional, contractile cardiomyocytes.

The contractile cycle of cardiomyocytes shown in FIGS. 1 and 2 (adaptedfrom Donald M. Bers, “Cardiac excitation-contraction coupling,” Nature,Vol 415, pp: 198-205, 2002) is divided into an abrupt shortening phase(systole), induced by a rapid rise in the intracellular calciumconcentration ([Ca²⁺]_(i)) due to calcium entry via voltage dependentL-type calcium channels and calcium-induced-calcium release from thesarcoplasmic reticulum (SR). Contraction is followed by relaxation ofthe cell (diastole) and the decline of [Ca²⁺]_(i). Decline of[Ca²⁺]_(i), during diastole is controlled primarily by re-sequestrationof calcium back into the SR by SERCA2, an ATPase associated with the SRmembrane.

Calcium transients have been recorded from cardiomyocytes derived fromembryonic stem cells (both from mouse and from human). For example,calcium transients in fura-2-loaded cardiomyocytes derived from humanembryonic stem cells (hESC-CM), were observed in spontaneouslycontracting embryoid bodies [K. Dolnikov, et al., “Functional Propertiesof Human Embryonic Stem Cell-Derived Cardiomyocytes,” Ann. N.Y. Acad.Sci. 1047, pp: 66-75, 2005]. hESC-CMs cultured on mousevisceral-endoderm like cells showed similar calcium transients andspontaneous contractions at 0.6 to 1.5 Hz [Mummery et al.,“Differentiation of Human Embryonic Stem Cells to Cardiomyocytes,”Circulation. 107, pp: 2733:2740, 2003]. Fluo-4 (a fluorescent dye) hasbeen used to record calcium transients from murine ESCMs, whichexhibited spontaneous contraction rates of approximately 1 Hz [Grey, etal., “Fine-tuning in Ca²⁺ homeostasis underlies progression ofcardiomyopathy in myocytes derived from genetically modified embryonicstem cells,” Human Molecular Genetics 14(10), pp: 1367-1377, 2005].Consistent with the wide spread observation of spontaneous beating inexperiments designed to elicit the appearance of ESCMs, myocytes with“pace-maker” activity have been observed in contracting cell clusters,serving to drive the contractions of neighboring cells [Mery et al.,“Initiation of Embryonic Cardiac Pacemaker Activity by Inositol1,4,5-Trisphosphate-dependent Calcium Signaling,” Mol Biol Cell, 16(5),pp: 2414-23, 2005]. These studies indicate that ESCMs typically exhibitcalcium transients and contractile characteristics similar to neonatalrat ventricular myocytes (NRVMs), rather than adult cardiomyocytes.

Automated high content screens allow for a wealth of information to begathered from a given experimental study. If the hardware andcontrolling software are present, researchers may be able to fine tunehardware performance to directly match their experimental needs.Unfortunately, this is not always the case and researchers are usuallyforced to either tweak their experimental design or are required tobuild customized tools to conduct their experiments. Thus, it would beextremely desirable to be able to record Ca⁺⁺ transients on acell-by-cell basis in a manner that is easily integrated into currentlaboratory setups. A desirable Calcium Transient Image Cytometer (CTIC)would interface easily into available high content microscopyworkstations which already perform multi-well plate scanning and imageacquisition, to enable video burst acquisition and analysis of calciumtransients in a fully automated (high throughput screening) mode. It isdesirable that the CTIC electrically stimulate (or pace) the cells,record the resulting Ca⁺⁺ transients from cells in microtiter plates(e.g., with 96 wells), and automatically quantify characteristics suchas the duration of the Ca⁺⁺ waves on a cell-by-cell basis in a fullyautomated manner on large scale screens (e.g., tens to hundreds ofthousands of compounds).

SUMMARY

A method, system, and instrument for automatically measuring transientactivity in cells uses image time sequences to identify transients incells. Preferably, the transient activity is stimulated or provoked insynchronism with acquisition of the image time sequences. A cell mask isapplied to each image of an image time sequence in order to localize thetransient activity with respect to each cell. Localization enablescell-by-cell analysis of properties of the transient activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a model of the cardiomyocyte contractile mechanismand FIG. 2 is a plot of the cardiac contractile cycle per the model ofFIG. 1.

FIG. 3 is a sequence of images with the same field of view thatillustrates a method for obtaining segmented cell masks from a videostream constituted of a time-series sequence of magnified images.

FIG. 4 data table illustrating storage of data obtained from a videostream constituted of a time-series sequence of magnified images usingcell masks.

FIG. 5 is a plot of transient curves made using data obtained from avideo stream constituted of a time-series sequence of magnified imagesusing cell masks.

FIG. 6 is a partially schematic illustration of an instrument foracquiring a time-series sequence of magnified images from a multi-wellplate or dish.

FIG. 7 is a block diagram of a system for performing a computer-executedmethod of automated transient image cytometry.

FIG. 8 is a flow chart illustrating a software program for performing acomputer-executed method of automated transient image cytometry.

FIG. 9 is a flow chart illustrating an image acquisition phase of thesoftware program of FIG. 8.

FIG. 10 is a flow chart illustrating an image segmentation phase of thesoftware program of FIG. 8.

FIG. 11 is a flow chart illustrating a cytometric analysis phase of thesoftware program of FIG. 8.

FIG. 12 is an illustration of a tangible medium of storage of a softwareprogram for operating an automated transient image cytometry system toperform a computer-executed method of automated transient imagecytometry.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

in order to obtain information about a transient event (like thecontractile cycle of cardiomyocytes) on a cell-by-cell basis in anautomated image cytometry system, a video stream constituted oftime-sequenced magnified images is acquired and an averaged image isobtained from averages of all images in the video stream. The averagedimage is segmented to obtain a cell periphery mask. A set of transientdata for each cell is obtained by applying the mask to each image of thevideo stream. For example, but without limiting the scope ofapplication, presume a video stream of magnified images is obtainedwhich is constituted of a time-series sequence of frames containingmagnified images of the same field of view showing cells marked with adye bound to elements active in a transient event. For example, presumethat the time-series sequence includes magnified images of fluo-4 loadedcardiac myocytes. With reference to FIG. 3, the time-series sequence offrames includes images A and B, among other images. Image A is a rawimage of NRVMs prior to stimulation of a Ca⁺⁺ transient by applicationof an electrical signal (low Ca⁺⁺, dim fluo-4). Image B is an image withthe same field of view as Image A, but later in time, immediately afterstimulation, when Ca⁺⁺ is high and fluo-4 is bright. All of the imagesin the video stream are averaged to obtain the averaged image denoted asImage C. Using known algorithms, cell periphery masks are segmented fromthe averaged image. The segmented masks are illustrated in Image Dwherein each cell mask is labeled with a unique gray-scale intensity forillustration of this description.

Once a cell periphery mask is generated, it is applied to each image inthe time-series sequence of images at the location of the cell itrepresents and the fluo-4 intensity within the mask is measured andrecorded. As per FIG. 4, showing tabular recording of intensity data forillustration only, each row in the table represents a frame (or image)in the video sequence where an image is obtained and each columnrepresents a cell. Thus, each data entry in a column represents thefluo-4 intensity (I) of the corresponding cell at a particular time of aCa⁺⁺ transient.

The intensity data points for a cell may be processed by known means toyield corresponding transient magnitude points which, in turn, may beused to plot a Ca⁺⁺ transient for the cell. Transients plotted usingsuch data points are shown in FIG. 5 for a control NRVM and for a NRVMmaintained in 100-nM thyroid hormone. The transients are normalized totheir minimum (0%) and maximum values (100%). Kinetic parameters whichmay be derived from the transients include Full Width at Half Max (FWHM)which is the time required for progression from the 50% point on theupstroke to 50% point on the downstroke, T_(up), which is the time fromthe 50% point to 100% on the upstroke, and T_(decay), which is the timeperiod from the 100% point to the 50% point on the decay phase.

An instrument illustrated in the schematic block diagram of FIG. 6 maybe used to acquire one or more video streams for processing images asillustrated in FIG. 3. The instrument 50 includes a robotic invertedmicroscope 51 outfitted for fluorescent microscopy. The microscope 51has an objective 52 and a mechanical stage 54 adapted to support amulti-well plate or dish. Conventional stage control mechanizationcauses the mechanical stage 54 to be moved in three dimensions. Thestage control mechanization causes the mechanical stage 54 to move in apredetermined pattern to bring wells of a multi-well plate or dish intoposition in the field of view of the microscope objective 52; when awell is positioned in the field of view of the objective 52, the stagecontrol mechanization moves the stage vertically for autofocus; theautofocus position is held while a video clip is obtained. Themechanical stage 54 is conventionally adapted to support a multi-wellplate or dish for being moved with respect to the field of view of theobjective 52. Such a plate may be one constructed with a transparentbottom so that the contents of its wells are visible to the objective52. For example, a glass-bottomed 96-well plate 55 may be used.

When transient activity analysis requiring stimulation of well contentsis to be conducted, the instrument of FIG. 6 may be further adapted toaccommodate the requirement. Such accommodation may support stimulationof well contents by light, pressure, electrical potential, material, andso on. In addition, a multi-well plate may be adapted to couple suchstimulation to its wells. For example, it may be desirable to stimulatecells by application of an electrical voltage. In this case, theinstrument of FIG. 6 may be adapted by provision of a coupling electrodeassembly 56 mounted on a moveable arm that moves the coupling electrodeassembly vertically with respect to the objective 52. Controlmechanization for positioning the coupling electrode assembly 52 may beconventional. That is to say, a control mechanization for positioningthe moveable arm to which the coupling electrode assembly 52 is mountedmay include, for example, a motor and a control routine for operatingthe motor to move the arm when the coupling electrode assembly 52 is tobe raised or lowered. The coupling electrode assembly 56 includeselectrically conductive electrodes 57 and 58 disposed in a spacedparallel relationship. A stimulator 59 applies a voltage across theelectrodes 57 and 58 and a polarity reverser 60 reverses the polarity ofthe voltage. In some aspects, the stimulator 59 and polarity reverser 60are constructed to operate in response to a controller 62 by causing theelectrodes 57 and 58 to deliver one or more shaped stimulus signals. Insome aspects, the shaped stimulus signals may be pulses (preferablyrectangular in shape) of electrical voltage.

The instrument of FIG. 6 may couple electrical stimulation to thecontents of wells in a multi-well plate or dish by moving the electrodes57 and 58 into and out of each well positioned in the field of view ofthe objective 52. However, the electrodes might have to be cleaned afterbeing withdrawn from a well so as not to contaminate the contents of thenext well. According to an alternate embodiment, each well may beprovided with a set of electrodes that are adapted to couple to theelectrodes 57 and 58 so that no set of electrodes enters more than asingle well. This solution is illustrated in FIG. 6 by a lid for amulti-well plate or dish. In this regard, a lid 70 seats on the top ofthe glass-bottomed 96-well plate 55. The lid 70 has multiple pairs oflid electrodes 72 mounted to it, each pair being positioned to extendinto a respective well when the lid 70 is seated on the top of the plate55. The lid electrode pairs 72 extend fully into their respective wellsand protrude above the lid 70; preferably, they are flexible,electrically conductive elements and may be retained in place at leastpartially by spring loading. The electrodes of each pair of lidelectrodes 72 are retained by the lid in the spaced parallelrelationship of the electrodes 57 and 58 so that when a well ispositioned in the field of view of the objective 52, the electrodes 57and 58 are longitudinally aligned with the lid electrode pair for thatwell. Lowering the coupling electrode assembly 56 carries the electrodes57 and 58 into electrical contact with the lid electrode pair of thepositioned well; raising the assembly carries the electrodes 57 and 58out of contact and permits the plate 55 to be repositioned. In someaspects, the electrodes 57 and 58 may include respective end receptacles57A and 58A shaped to facilitate coupling to lid electrode pairs. Forexample, the end receptacle of each coupling electrode may be shapedwith a wide mouth and an internal tapered cone to capture and makeelectrical contact with flexible and spring-loaded lid electrode tipsthat protrude above the lid.

FIG. 7, which is meant for example and not for limitation, illustratesan automated image cytometry system adapted to perform automatedtransient image cytometry. The system is adapted by addition ofprogramming for the video stream processing illustrated in FIG. 3 to theautomated image cytometry programming and by addition of theinstrumentation illustrated in FIG. 6. The system performs acomputer-executed method of automated transient image cytometryaccording the Detailed Description and which may be implemented in partin a software program, for example, a program written in the C++ and/orJava programming languages, and a counterpart system may be a specialpurpose computer system designed to execute the method. Of course, themethod and the programmed computer system may also be embodied in aspecial purpose processor provided as a set of one or more chips.Further, there may be a program product constituted of a program ofcomputer or software instructions or steps stored on a tangible articleof manufacture that causes a computer to execute the method. Thetangible article of manufacture may be constituted of one or more realand/or virtual data storage articles.

In FIG. 7 the automated instrumentation system is constructed andprogrammed to perform automated image processing, image data management,and image data analysis operations of HCS and/or HTS systems. Forexample, the instrumentation system may be, or may reside in, or may beassociated with a microscopy system 100 including the microscope 51 withthe motorized, automatically moveable stage 54 on which a carrier withbiological material may be disposed for observation by way of themicroscope 51. The carrier may be the multi-well plate 55 discussedabove whose wells are disposed in a two dimensional array. For example,and without limitation, the multi-well plate 55 may be a ninety-six wellmicro-titer plate in each well of which there is biological materialthat has been cultured, activated, fixed, and stained. A light source118 provides illumination for operation of the microscope 51 by way ofan optical filter 120 and a fiber optic cable 122. The moveable stage112 may be intermittently or continuously moved to enable theacquisition of one or more video streams each constituted of atime-series sequence of magnified images. Images in the field of view ofthe objective 52 are directed by mirrors and lenses to a high-resolutiondigital camera 126. The camera 126 obtains and buffers video streams andtransfers them on an interface 127 to a processor 128. The interface 127may be, for example and without limitation, a universal serial bus(USB).

The frames of each video stream are constituted of digital images whichmay be provided in some standard format comprising an N×M array ofpixels to the processor 128. The processor 128 receives the digitalimages and stores them in image files. The digital images are processedby the processor 128 and output digital images may be provided by theprocessor 128 for display on an output device with a display 130.

With further reference to FIG. 7, the processor 128 may be a programmedgeneral purpose digital processor having a standard architecture, suchas a computer work station. The processor 128 includes a processing unit(CPU) 140 that communicates with a number of peripheral devices by wayof a bus subsystem 142. The peripheral devices include a memorysubsystem (MEMORY) 144, a file storage subsystem (FILE) 146, userinterface devices (USER) 148, an input device (INPUT) 149, and aninterface device (INTERFACE) 150. It is not necessary that the processor128 be connected directly to the microscope 51; it may receive imagesproduced by the camera 126 from a portable storage device, or by way ofa local or wide area network. For example, images may be transported tothe processor 128 over the internet.

The bus subsystem 142 includes media, devices, ports, protocols, andprocedures that enable the processing unit 140 and the peripheraldevices 144, 146, 148, 149, and 150 to communicate and transfer data.Instrumentation 156 such as is illustrated in FIG. 6 may also be coupledto the bus subsystem 142 by a conventional instrumentation interface.The bus subsystem 142 provides generally for the processing unit andperipherals to be collocated or dispersed

The memory subsystem 144 includes read-only memory (ROM) for storage ofone or more programs of instructions that implement a number offunctions and processes. One of the programs is an automated imageprocess for processing a magnified image of biological material toidentify one or more components of an image. The memory subsystem 144also includes random access memory (RAM) for storing instructions andresults during process execution. The RAM is used by the automated imageprocess for storage of images generated as the process executes. Thefile storage subsystem 146 provides non-volatile storage for program,data, and image files and may include any one or more of a hard drive,floppy drive, CD-ROM, and equivalent devices

The user interface devices 148 include interface programs and input andoutput devices supporting a graphical user interface (GUI) for entry ofdata and commands, initiation and termination of processes and routinesand for output of prompts, requests, screens, menus, data, images, andresults.

The input device 149 enables the processor 128 to receive digital imagesdirectly from the camera 126, or from another source such as a portablestorage device, or by way of a local or wide area network. The interfacedevice 150 enables the processor 128 to connect to and communicate withother local or remote processors, computers, servers, clients, nodes andnetworks. For example, the interface device 150 may provide access to anoutput device 130 by way of a local or global network 151.

FIGS. 8-11 illustrate a software program containing instructions foroperating a system such as that shown in FIG. 7 to perform acomputer-executed method of automated transient image cytometry. Anoverview of an automated transient image cytometry method is seen inFigure where, at 200, the system initiates method at 200. The systemacquires one or more video streams of cells exhibiting transientactivity from one or more wells in a multi-well pate or dish at 202. At204, at least one cell periphery mask is generated for each well where avideo stream is acquired. Cytometric analysis using data generated withthe cell periphery masks is performed at 206, and the method terminatesat 207.

The acquisition of one or more video streams in step 202 of FIG. 8 isillustrated in more detail in FIG. 9, which is explained with referenceto the instrument of FIG. 6. At 220, the mechanical stage 54 is moved toplace a well in the field of view of the objective 52. At 222, theelectrodes 57 and 58 are lowered to provide a stimulus signal to thewell. Using the stained nuclei of cells in the well, an autofocusprocess adjusts the mechanical stage 54 vertically to a best focusposition where the contents of the well are best focused by theobjective 52. Then, at 224, with best focus established, a first filteris utilized to allow the objective 52 to image cell nuclei and a nuclearimage is acquired and stored. At 225, using a second filter to establisha channel of interest, the camera shutter is operated to startacquisition of a video stream constituted of a time-series sequence ofmagnified images of objects in the well's contents. In this regard thechannel of interest may be associated with or determined by the color ofobjects viewed through the second filter. Immediately thereafter, at226, a stimulus signal is applied to the well in order to initiatetransient activity in cells contained in the well. A time-seriessequence of images of the cells, including those cells exhibitingtransient activity, is captured in the ongoing video stream. After apredetermined amount of time sufficient to capture the transient event,the camera shutter is operated to cease acquisition. At 228, theelectrodes are retracted and the time-series sequence of images isstored for later processing. At 230, as long as there is another fromwhich to acquire a video stream, the process repeats; otherwise, theprocess ends.

After acquiring a time-series sequence of images of the contents of awell, the system initiates cell mask generation as per FIG. 10. At 240,a time-series average image (per FIG. 3) is obtained using the channelof interest. At 242, the nuclear image for the well is segmented and thesegmented nuclear image is used with the time-series average image toobtain cell masks at 244. Generation of cell masks may be done using aknown technique such as a watershed algorithm.

After acquiring cell masks for a time-series sequence of images of thecontents of a well, the system initiates cytometric analysis as per FIG.11. At 250, the cell masks are applied to each frame of the time-seriessequence of images and total and average intensity magnitudes for eachcell are obtained at 252 using the cell masks. At 254, the system mayproduce a plot showing average channel intensity versus time for eachcell in the well as illustrated in FIG. 5.

Methods and apparatuses for practicing these teachings may beconstituted in whole or in part of a program product with acomputer-readable storage medium, network, and/or node that enables acomputer, a processor, a fixed or scalable set of resources, a networkservice, or any equivalent programmable real and/or virtual entity toexecute an automated transient image cytometry process as described andillustrated above. The program product may include a portable mediumsuitable for temporarily or permanently storing a program of softwareinstructions that may be read, compiled and executed by a computer, aprocessor, or any equivalent article. For example, the program productmay include a programmed CD such as is seen in FIG. 12, or anetwork-accessible site, node, center, or any equivalent article.

INDUSTRIAL EXAMPLES

Cultured NRVMs were utilized according to the description andillustrations above to obtain the images of FIG. 3 and the plots of FIG.5. These can be prepared quickly (as opposed to the more lengthyculturing required for ESCMs). The cultured NRVMs respond to electricalstimulation a Ca⁺⁺ transient accompanied by a contractile-twitch. Thecontractile calcium transients are transient elevations in intracellularcalcium, which can be tracked via use of intracellular fluorescentcalcium indicators (e.g., Fluo-3 and Fluo-4) and digital fluorescencemicroscopy technology as represented by the EIDAQ 100 (Q3DM, San Diego,Calif.) modified to obtain and process video streams as illustrated inFIGS. 3 and 4. An instrument designed to electrically stimulate, record,and quantify contractile calcium transients from NRVMs cultured in96-well dishes, should be capable of recording electrically stimulatedcontractile calcium transients in cultures of embryonic stem cells toprovide a definitive readout of the stage of differentiation towardscardiomyocytes. That is, we hypothesize that different stages ofdifferentiation will be characterized by measurement differences in theCa⁺⁺ transients of these beating cells. The instrument will also likelybe useful for testing compounds on NRVMs (or cardiac myocytes from othersources) in a high throughput manner for potential negative or positiveinotropic effects, or for arrhythmic effects.

The NRVMs were cultured in 96-well dishes (Nunc coverslip-glass-bottomplates) pretreated with 1% gelatin that was cross linked withglutaraldehyde to improve cell adherence.

Sensitivity to intracellular calcium kinetics is a desirable goal forautomated calcium transient image cytometry because it is desirable todistinguish healthy contractile ESCMs from background non-contractilestem cells. To test this sensitivity, contractile calcium transientswere quantified in two populations of NRVMs: NRVMs exposed to controlmedia vs. media supplemented with 100-nM thyroid hormone (T3), which isknown to increase the expression of SERCA2, leading to more rapidcalcium re-uptake, and, overall accelerated kinetics of the contractilecalcium transients (FIG. 5). Thus, for all NRVMs preparations, half ofthe wells contained control medium (DMEM:F12 with 0.25% FBS) and theremainder included the same media supplemented with T3. The cells weremaintained 48 to 72 hrs prior to scanning. On the day of scanning, thecells were incubated with a 3 μM solution of the acetyoxymethylesterform of an intracellular calcium indicator (either Fluo-4 AM or Fluo-3AM) in the base media for 25 minutes. The plasma membrane permeable tothe acetoxymethylester forms of Fluo-3 and Fluo-4, which are modifiedintracellularly by esterases to an impermeable form. The loadingsolution was also supplemented with 200 ng/ml Hoechst 33342 to label DNAwithin the nuclei. This concentration of Hoechst provides adequate labelof the nuclei and was non-toxic under these conditions. Thus, thelabeling procedure introduced a green-fluorescence channel fluorescentcalcium indicator into the cytoplasm of the NRVMs (Fluo-4) and ablue-fluorescence channel nuclear stain (Hoechst). The nuclear channelwas used for autofocus at each well; additionally, images obtained ofthe nuclei provided information regarding cellular position that washelpful in assigning boundaries between cells in the segmentationprocedure applied to the green-channel images. Following loading, cellswere placed in 150 μl of HEPES-buffered physiological saline (Tyrodessolution) supplemented with 5-mM glucose and 2-mM calcium chloride.

This example is merely illustrative and is not intended to limit theapplication of the principles set forth above to automated measurementand analysis of calcium transients in cardiomyocytes. Other applicationsmay include stem cell, cardiac myocytes derived from stem cells (bothembryonic stem cells and induced pluripotent stem cells), neurons,pancreatic beta-cells, adrenal cells, and PC12 cells (which are neuronallike, but derived from the adrenals). In addition to electric signals,stimuli to provoke transient response may include chemicals (e.g.,potassium chloride), calcium channel activators (e.g., BayK8644),pressure, light, and sound. Other transients to be measured may includemembrane potential across the plasma membrane, membrane potential acrossthe mitochondrial membrane, intracellular sodium, and intracellularpotassium.

Exemplary systems which can be used in the practice of the detaileddescription given above include the high content screening system, theEIDAQ 100 (Q3DM, San Diego, Calif.) which is functionally identical tothe later-released Beckman Coulter IC 100. The EIDAQ 100 includes: 1) aninverted epifluorescence microscope (Nikon Eclipse TE2000-U), 2) anintensity-feedback stabilized 100 W Hg arc lamp developed by Q3DM, 3)excitation and emission filter wheels, 4) a motorized stage with XY-axescontrol, 5) a piezoelectric Z-axis control for fast, precise autofocus,and 6) the software sold by Vala Sciences, Inc. under the CytoShop™brand. The emission light path on the EIDAQ 100 is split by a Nikonmultimode module to the autofocus and scientific grade CCD cameras. TheEIDAQ 100's scientific grade camera was removed from the optical pathbut left powered on and connected to allow normal operation of CytoShop™software.

Instrumentation according to FIG. 6 was built using a PC running WindowsXP and control software programmed in Labview 8.0, (NationalInstruments, Austin, Tex.), a National Instruments data acquisition I/Oboard, the NI-PCI-6251, a stimulator/electrode assembly (lowered andraised using a computer-controlled Sutter Instruments (Novato, Calif.)micromanipulator, the MP-285), a Grass Technologies (West Warwick, R.I.)S48 square pulse stimulator, and a high speed scientific-grade CCDcamera. Back- and front-illuminated EMCCD cameras were tested includingthe iXon DU-897 from Andor (South Windsor, Conn.), the ImageEM C9100-13from Hamamatsu (Hamamatsu, Japan), and the QuantEM fromRoper/Photometrics (Tuscon, Ariz.).

The scope of patent protection afforded the novel tools and methodsdescribed and illustrated herein may suitably comprise, consist of, orconsist essentially of any combination of the elements described above.Further, the novel tools and methods disclosed and illustrated hereinmay suitably be practiced in the absence of any element or step which isnot specifically disclosed in the specification, illustrated in thedrawings, and/or exemplified in the embodiments of this application.Moreover, although the invention has been described with reference topresently preferred embodiments, it should be understood that variousmodifications can be made without departing from the spirit of theinvention.

The invention claimed is:
 1. An instrument for obtaining a time-seriessequence of magnified images of transient activity in cells, comprising:an adjustable mechanical stage to position a well of multi-well plate;means for applying a stimulation signal in the well; a microscopeobjective with a field of view containing the well; an autofocus controlmechanization operative to adjust the mechanical stage vertically to abest focus position where the contents of the well are best focused bythe objective; and, a camera optically coupled to the microscopeobjective and operative to acquire a time-series sequence of magnifiedimages of the contents.
 2. The instrument of claim 1, wherein the meansfor applying a stimulation signal includes means for applying one oflight, pressure, electrical potential, and material to the cells.
 3. Theinstrument of claim 1, wherein the means for applying a stimulationsignal includes means for coupling an electrical voltage to the cells.4. The instrument of claim rein the means for coupling an electricalvoltage to the cells includes electrodes.